PHYTOCHEMISTRY Phytochemistry 68 (2007) 591–595 www.elsevier.com/locate/phytochem
a-Glucosidase inhibitory pentacyclic triterpenes from the stem bark of Fagara tessmannii (Rutaceae) Luc Meva’a Mbaze a, Herve Martial P. Poumale b,c, Jean Duplex Wansi a,*, Jean Alexandre Lado a, Shamsun Nahar Khan d, Muhammad Choudhary Iqbal d, Bonaventure Tchaleu Ngadjui c, Hartmut Laatsch b b
a Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon Department of Organic and Biomolecular Chemistry, University of Go¨ttingen, Tammannstrasse 2, D-37077 Go¨ttingen, Germany c Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon d H.E.J Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
Received 8 September 2006; received in revised form 11 December 2006 Available online 31 January 2007
Abstract In addition to fatty acids, a mixture of sterols (b-sitosterol, stigmasterol, campesterol and stigmastanol), lupeol, arctigenin methylether, sesamin, vanillic acid (1), 2,6-dimethoxy-1,4-benzoquinone (2), betulinic acid and two pentacyclic triterpene acetates were isolated from Fagara tessmannii Engl. They were identified as 3b-acetoxy-16b-hydroxybetulinic acid (3a) and 3b,16b-diacetoxybetulinic acid (3b), and their structures were established using 1 and 2D NMR spectra and by comparison with published data. Two derivatives of the compounds were prepared. Some isolated compounds were evaluated for their antifungal and antibacterial activities. Compounds 1 and 3a showed significant inhibition of a-glucosidase. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Fagara tessmannii; Rutaceae; Pentacyclic triterpene; Antifungal; Antibacterial; a-Glucosidase inhibition
1. Introduction Fagara tessmannii Engl. (Rutaceae) (Syn. Zanthoxylum tessmannii) is a shrub of the African rainforests in South-West, Centre, South and East provinces in Cameroon. In the traditional medicine, it is used locally for the treatment of tumors, swellings, inflammation and gonorrhoea (Raponda-Walker and Sillans, 1961). The root bark of this plant is used in West Africa in traditional medicine as a toothbrush (Kerharo and Adam, 1971). Previous phytochemical studies of F. tessmannii collected at three different localities in Cameroon resulted in the isolation of alkaloids, lignans, triterpenes and steroids (Ayafor et al., 1984). The widespread use of F. tessmannii in indigenous
*
Corresponding author. Tel.: +237 781 77 31. E-mail address:
[email protected] (J.D. Wansi).
0031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.12.015
medicine for different ailments, as well as the antifungal and antioxidant activities exhibited by the genus (Chaaib et al., 2003), justified further attempts to isolate and identify active compounds. In this paper, we report the isolation and structure elucidation of two new pentacyclic triterpene acetates, and their biological activities. 2. Results and discussion The stem bark of F. tessmannii was extracted with MeOH. The extract was submitted to repeated column chromatography and preparative TLC (PTLC) to afforded fatty acids, a mixture of sterols (b-sitosterol, stigmasterol, campesterol and stigmastanol), lupeol, arctigenin methylether, sesamin, betulinic acid, vanillic acid (1), 2,6-dimethoxy-1,4-benzoquinone (2), and two new pentacyclic triterpene acetates (3a, 3b). The 1H and 13C NMR, and
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MS of the known compounds were consistent with those reported in the literature. Compound 3a was obtained as a white powder from the hexane/ethyl acetate (7:3) extract B. The (+)-ESI HR mass spectrum indicated a pseudomolecular ion at m/z 532.39963 [M + NH4]+, corresponding to a molecular formula C32H54NO5. The IR spectrum showed a broad signal at t 3415 cm1 indicating free hydroxyl group, and an ester signals at t 1724 (C@O) and 1247 (C–O) cm1. Three absorptions at t 2947, 1641 and 888 cm1 indicated a vinylidene group (@CH2), characteristic of lup-20(29)-ene (Roitman and Jurd, 1978). The 1H NMR data indicated that compound 3a is a pentacyclic triterpenoid (Cheung and Williamson, 1969) with five methyl groups between d 0.81–1.12 (s, 3H each), an olefinic methyl at d 1.70, an acetyl methyl at d 2.09, seven methine and nine methylene groups between d 1.12 and 2.20. A methine signal at d 3.20 (ddd, J = 5.2, 10.8, 12.8 Hz) was attributed to the proton at position C-19, and one proton signal at d 3.74 (dd, 1H, J = 4.4 and 11.3 Hz) indicated the presence of oxygen and is characteristic of an equatorial (b) orientation at position C-16. A proton signal at d 4.61 (t br, 1H) is due to the presence of an acetoxy group, two olefinic signals at d 4.64 (d br, 1H, J = 1.3 Hz, Hb-29) and 4.76 (d br, 1H, J = 1.3 Hz, Ha-29) indicated an exomethylene group. The 13C NMR spectrum (Table 1) of compound 3 revealed the presence of 30 carbon atoms, which were in accordance with the proton data, and additionally a carboxylic acid signal at d 177.9 and an acetate carbonyl at d 171.0. The carbon atoms at d 149.4 and 110.0 are characteristic for the carbons 20 and 29 of lup-20(29)-ene (Gunasekera et al., 1982). These data indicated that compound 3a might be a lupan-type triterpene. The fragment at m/z 189 supported the presence of lup-20(29)-ene (Kumar et al., 1985). In the HMBC spectrum (Fig. 1), correlations between the H-3 signal and carbons 1, 2, 4, 5 and 1 0 , between the proton H-16 and carbons 14, 15, 17, 18 and 28, and finally the correlation of H-19 with C-18, 20, 21, 22, 27 and 29 as well as the close similarity with the shifts of 16b-hydroxybetulinic acid (3e) (Table 1) indicated that compound 3a is 3b-acetoxy-16b-hydroxybetulinic acid, which is described here for the first time. Compound 3b was isolated as amorphous solid from the same fraction B eluted with hexane/ethyl acetate (7:3). The (+)-ESI HR mass spectrum indicated a pseudomolecular ion at m/z 574.41021 [M + NH4]+, corresponding to the molecular formula C34H56NO6. Comparison of the spectral data of compound 3a and 3b indicated an additional acetate group in 3b, which was confirmed by the peak at m/z 496 [MCH3CO–H2O]+. The IR spectrum revealed the expected ester signals at t 1729 cm1 (C@O) and 1246 cm1 (C–O). The 1H NMR spectra of 3a and 3b were nearly superimposable, the only difference being the presence of a further acetate group at d 2.08 and the down-field shift of the H-16 signal from d 3.74 in 3a to d 5.05 in 3b. The main difference
Table 1 13 C NMR of compounds 3a, b, c and d (75, 125 and 150 MHz, CDCl3) in comparison with 16b-hydroxybetulinic acid (3e) C Nr
3a
3b
3c
3d
3e
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 10 20 100 200 CH3O
36.6 22.8 78.4 39.3 50.2 18.0 35.1 40.9 49.8 37.1 20.5 25.1 37.8 44.2 34.1 76.1 60.7 48.9 46.8 149.4 30.7 33.9 27.8 15.9 16.1 16.3 21.4 177.9 110.0 19.5 171.0 21.6 – – –
36.6 28.0 78.3 39.0 50.2 21.3 34.9 41.2 49.9 37.0 21.7 24.6 38.1 44.2 37.5 77.1 59.5 49.5 47.3 148.8 33.9 34.6 27.7 15.9 18.0 18.8 15.9 176.9 110.6 20.5 170.3 21.4 170.9 22.8 –
36.6 22.8 78.3 37.1 50.2 19.3 35.2 40.9 49.7 33.9 21.7 25.0 38.2 44.1 39.7 76.3 61.4 49.3 47.5 149.5 30.9 34.0 27.8 16.2 18.0 15.9 15.8 176.8 110.1 20.6 170 21.4 – – 51.6
36.6 24.5 78.3 34.6 50.2 18.6 33.4 41.2 49.5 30.5 21.7 22.8 37.1 44.0 34.6 76.8 59.5 49.5 47.7 151.3 27.8 34.2 29.5 15.9 18.0 15.9 15.9 173.5 110.7 20.6 170.6 21.4 170.9 21.3 51.5
38.9 28.7 77.1 39.3 56.0 18.6 34.3 41.6 50.1 37.9 20.8 25.0 38.4 44.2 39.9 75.9 62.1 49.0 48.6 149.9 32.3 36.5 28.5 16.2 16.5 16.6 16.1 178.1 110.4 19.4 – – – – –
in the 13C NMR spectrum (Table 1) was the presence of an additional ester carbonyl signal at d 170.9. The analysis of the data and comparison with those of 3a and 16b-hydroxybetulinic acid (3e) indicated that 3b is 3b,16b-diacetoxybetulinic acid, which is described here for the first time. Methylation of compound 3a with diazo methane yielded 3c (C33H52O5), and acetylation of 3c gave 3d (C35H54O6). Their structures were confirmed by EI, HRESIMS, 1H and 13C NMR spectra (see Section 3). The antifungal and antibacterial activities of 2 and 3a, b, c and d were determined using the agar diffusion method with 9 mm paper disks loaded with 40 lg of each compound isolated from this plant. Only 2,6-dimethoxy-1,4benzoquinone (2) showed activities against Bacillus subtilis (20 mm inhibition diameter), Staphylococcus aureus (17 mm), Escherichia coli (14 mm), Streptomyces viridochromogenes (Tu¨ 57) (12 mm), Mucor miehei (12 mm), Chlorella vulgaris (10 mm) and Scenedesmus subspicatus (10 mm); 3b-acetoxy-16b-hydroxybetulinic acid (3a) showed weak activities against Bacillus subtilis (13 mm) and Escherichia coli (11 mm), and 3b,16b-diacetoxybetuli-
L.M. Mbaze et al. / Phytochemistry 68 (2007) 591–595
potent and selective inhibition of yeast a-glucosidase than against the other enzymes (Table 2). Compound 3a showed significant higher inhibition of yeast a-glucosidase than deoxynojirimycin (425.6 ± 8.1 lM) and acarbose (780.0 ± 0.1 lM).
Structures of compound 1, 2, 3a, b, c, d and e
OH
O
O O
O
3. Experimental section
OMe OH
O
1
H
30
29
20
H
1
18
22
3
COOR 28
17 14
9
16
8
3
15
NMR spectra were measured on Varian Unity 300 (300.145 MHz) and Varian Inova 500 (499.876 MHz) spectrometers. ESI MS was recorded on a Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument). ESI HR mass spectra were recorded on A Bruker FTICR 4.7 T mass spectrometer. EI-MS spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV) with perfluorkerosene as reference substance for HREI MS. IR spectra were recorded on a Perkin-Elmer 1600 Series FT-IR spectrometer from films. Flash chromatography was carried out on silica gel (230–400 mesh). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co.).
21
19
26
10
a
13
11 25
2
OR
27
4
1
RO
7
5 6
23
24
3a 3b 3c 3d 3e
R1 Ac Ac Ac Ac H
3.1. Materials and methods
2
b
12
2
593
R2 H Ac H Ac H
R3 H H Me Me H
3.2. Plant material The stem bark of F. tessmannii Engl. was collected in April 2005 at Limbe, South-West Cameroon. A specimen has been deposited in the National Herbarium, Yaounde´, Cameroon (Ref. No. 1490/SRFK). COOH
O
4. Extraction and isolation
O
1' 2'
O
O
1''
2''
Fig. 1. Selected HMBC correlation in compound 3b. Respective correlations were found also in compound 3a.
nic acid (3b) showed weak activities against Bacillus subtilis (12 mm) and Candida albicans (14 mm). When tested again three common glycosidases, compounds 3a (7.6 ± 0.6 lM) and 1 (69.4 ± 0.8 lM), shown Table 2 Glycosidase inhibition of some isolated compounds Compound
3a 1 2 Arctigenin methylether Lupeol Mixture of steroids Sesamin
a-DGlucosidase (yeast)
b-D-Glucosidase (sweet almonds)
a-DMannosidase (jack bean)
IC50 ± SEM
IC50 ± SEM
IC50 ± SEM
7.6 ± 0.6 69.4 ± 0.8 900.0 ± 3.5 NI
397.6 ± 0.8 295.8 ± 0.5 NI NI
NI NI NI NI
NI NI
NI NI
NI NI
NI
NI
NI
NI, no inhibition at 800 lM concentration.
The powdered stem bark of Fagara tessmannii (1.5 kg) was extracted with MeOH at room temperature during 48 h. After removing the solvents by evaporation under reduced pressure, the crude extract (73.2 g) was chromatographed on silica gel. Using hexane/ethyl acetate of increasing polarity, a total of 125 sub-fractions (ca. 250 ml each) were collected and combined on the basis of TLC analysis leading to three main fractions A, B and C. Fraction A (15.0 g) was chromatographed on silica gel and eluted with a mixture of hexane/ethyl acetate of increasing polarity to yield fatty acids (104.0 mg) (Andersen and Gorbert, 2002), mixture of sterols (GC showed the presence of b-sitosterol, stigmasterol, campesterol and stigmastanol) (102.0 mg) (Morris et al., 1984) and lupeol (69.0 mg) (Razdan et al., 1996). Fraction B (7.0 g) was chromatographed on silica gel and eluted using hexane/ethyl acetate (7:3) to deliver betulinic acid (17.0 mg), 3b-acetoxy-16b-hydroxybetulinic acid 3a (11.0 mg), 3b,16b-diacetoxybetulinic acid 3b (4.0 mg), arctigenin methylether (6.0 mg) and sesamin (5.0 mg) (Ayafor et al., 1984). Fraction C (11.0 g) produced in the same way vanillic acid 1(12.5 mg) (Shchukin and Medvedeva, 1999) and 2,6-dimethoxy-1,4-benzoquinone (2) (15.0 mg).
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4.1. 3b-Acetoxy-16b-hydroxybetulinic acid (3a) White powder, Rf = 0.53 (CH2Cl2); m.p. 255–257 °C; IR (film): t = 3415, 2947, 2873, 1724, 1641, 1451, 1375, 1247, 1183, 1040, 1020, 975, 888 cm1; 1H NMR (300 MHz, CDCl3): d 4.76 (d br, 1 H, J = 1.3 Hz, Ha-29), 4.64 (d br, 1H, J = 1.3 Hz, Hb-29), 4.61 (t br, 1H, H-3), 3.74 (dd, 1H, J = 4.4, 11.3 Hz, H-16), 3.20 (ddd, 1H, J = 5.2, 10.8, 12.8 Hz, H-19), 2.15 (m, 2H, H-21), 2.13 (m, 2 H, H-15), 2.09 (s, 3H, Ac), 2.05 (m, 2H, H-12), 1.90 (m, 1H, H-18), 1.70 (s, 3H, H-30), overlapping multiplets at 1.72, 1.67, 1.62, 1.61, 1.60, 1.58, 1.50, 1.48, 1.40, 1.39, 1.38, 1.20, 1.10 (m, 15H, H-1, 2, 5, 6, 7, 9, 11, 13, 22), 1.10 (s, 3H, H-26), 0.98 (s, 3H, H-27), 0.87 (s, 3H, H-23), 0.83 (s, 3H, H-25), 0.82 (s, 3H, H-24); 13C NMR (125 MHz, CDCl3) (Table 1); EI MS (70 eV): m/z (%) 514 (M+, 5), 454 (6), 189 (11), 145 (5), 107 (7), 81 (10), 55 (13), 43 (100); (+)ESI HRMS: m/z 532.39963 ([M+NH4]+, calcd 532.39965 for C32H54NO5). 4.2. 3b,16b-Diacetoxybetulinic acid (3b) Amorphous solid, Rf = 0.84 (CH2Cl2); IR (film): t = 3426, 3077, 2944, 2874, 1729, 1644, 1453, 1374, 1246, 1182, 1026, 985, 964, 920, 888, 757 cm1; 1H NMR (300 MHz, CDCl3): d 5.08 (dd, 1H, J = 4.9 and 11.7 Hz, H-16), 4.78 (d br, 1H, J = 1.4 Hz, Ha-29), 4.62 (m br, 2H, Hb-29 and H-3), 2.78 (ddd, 1H, J = 5.4, 11.3 and 13.7 Hz, H-19), 2.40–2.12 (m, 4H, H-15 and H-21), 2.10 (s, 6H, H-2 0 and H-200 ), 1.98–1.70 (m, 3H, H-18 and H22), 1.68 (s, 3H, H-30), 1.65-1.12 (m, 15H, H-1, H-2, H5, H-6, H-7, H-9, H-11, H-12, H-13), 1.10 (s, 3H, H-25), 1.07 (s, 3H, H-26), 0.83 (s, 9H, H-23, H-24, H-27); 13C NMR (150 MHz, CDCl3) (Table 1); EI MS (70 eV): m/z (%) = 556 (M+, 11), 496 (50), 481 (10), 452 (8), 436 (42), 421 (10), 392 (13), 246 (11), 213 (15), 199 (27), 190 (38), 189 (100), 190 (44), 173 (28), 135 (44), 95 (44), 81 (39), 43 (69); (+)-ESI HRMS: 574.41021 ([M+NH4]+, calcd 574.41074 for C34H56NO6). 4.3. 3b-Acetoxy-16b-hydroxybetulinic acid methyl ester (3c) Compound 3a (5 mg), was dissolved in methylene chloride (2 ml) and an etherial diazomethane solution (2 ml) was added at 20 °C. Immediate evaporation to dryness gave 3c (5 mg, 97%) as an amorphous solid with Rf = 0.63 (CH2Cl2); 1H NMR (300 MHz, CDCl3): d = 4.90 (d br, 1H, J = 3.0 Hz, Ha-29), 4.75 (d br, 1H, J = 3.0 Hz, Hb29), 4.61 (t br, 1H, J = 1.7 Hz, H-3), 3.78 (s, 3H, MeO-), 3.60 (t br, 1H, J = 2.4 Hz, H-16), 2.99 (ddd, 1H, J = 5.3, 10.9 and 12.9 Hz, H-19), 2.15 (m, 1H, H-13), 2.08 (s, 3H, H-2 0 ), 2.06–1.80 (m, 4H, H-12, H-21), 1.70 (s, 3H, H-30), 1.68–1.11 (m, 16H, H-1, H-2, H-5, H-6, H-7, H-9, H-11, H-15, H-22), 1.08 (s, 3H, H-26), 0.92 (s, 3H, H-27), 0.88 (s, 3H, H-23), 0.83 (s, 6H, H-24, H-25);13C NMR (75 MHz, CDCl3) (Table 1); EI MS (70 eV): m/z (%) = 528
(M+, 13), 510 (100), 469 (10), 450 (30), 435 (9), 391 (9), 260 (10), 246 (13), 199 (24), 189 (38), 173 (11), 135 (18), 107 (18), 81 (20), 69 (17), 43 (20); (+)-ESI HRMS: 529.38875 ([M + H]+, calcd 529.38928 for C33H53O5), 551.3707 [M+Na]+, calcd 551.37122 for C33H52NaO5. 4.4. 3b,16b-Diacetoxybetulinic acid methyl ester (3d) 3-Acetoxy-16-hydroxybetulinic acid methyl ester (3c, 3 mg) was dissolved in pyridine (0.4 ml) and acetanhydride (0.9 ml). The solution was stirred for 6 h at 35 °C. Hydrolysis and usual work-up gave 3d (2 mg, 62%) as an amorphous solid with Rf = 0.81 (CH2Cl2); 1H NMR (300 MHz, CDCl3): d 5.1 (dd, 1H, J = 5.0 and 11.4 Hz, H-16), 4.70 (d br, 1H, J = 0.8 Hz, Ha-29), 4.60 (d br, 2H, Hb-29 and H3), 3.72 (s, 3H, MeO), 3.06 (m br, 1H, H-19), 2.50 (ddd, 1H, J = 5.2, 10.7 and 11.3 Hz, H-13), 2.44–2.13 (m, 4H, H-15, H-21), 2.10 (s, 3H, H-2 0 ), 2.03 (s, 3H, H-200 ), 2.00– 1.70 (m, 7H, H-1, H-12, H-18, H-22), 1.67 (s, 3H, H-30), 1.60–1.18 (m, 12H, H-2, H-5, H-6, H-7, H-9, H-11, H-21), 1.17 (s, 3H, H-26), 1.14 (s, 3H, H-27), 0.88 (s, 3H, H-23), 0.87 (s, 3H, H-24), 0.86 (s, 3H, H-25); 13C NMR (125 MHz, CDCl3) (Table 1); EI MS (70 eV): m/z (%) = 570 (M+, 2), 510 (100), 495 (6), 450 (23), 435 (6), 391 (8), 260 (10), 246 (12), 199 (18), 187 (35), 185 (18), 135 (12), 107 (13), 81 (18), 43 (79); (+)-ESI HRMS: 588.42587 ([M+NH4]+, calcd 588.42639 for C35H58NO6), 593.38126 ([M+Na]+, calcd 593.38179 for C35H54NaO6).
5. Biological activities 5.1. Antimicrobial assay Agar diffusion tests were performed in the usual manner (Maskey et al., 2002) with Bacillus subtilis and Escherichia coli (on peptone agar), Staphylococcus aureus (Bacto nutrient broth), Streptomyces viridochromogenes (M Test agar), the fungi Mucor miehei and Candida albicans (Sabouraud agar), and three microalgae (Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus subspicatus). Compounds were dissolved in an chloroform/MeOH (87:13) azeotrope and paper disks (Ø 9 mm) were impregnated with each 40 lg using a 100 ll syringe, dried for 1 h under sterile conditions and placed on the pre-made agar test plates. Bacteria and fungi plates were kept in an incubator at 37 °C for 12 h, micro algae plates for three days at room temperature in a day light incubator. The diameter of inhibition zones was measured. 5.2. Glycosidase inhibition The glycosidase inhibition assay was performed according to the slightly modified method of Oki et al. (1999). Activity of the compounds has been determined against a-D-Glucosidase (E.C. 3.2.1.20), b-D-Glucosidase (E.C. 3.2.1.21), and a-D-Mannosidase (E.C. 3.2.1.24),
L.M. Mbaze et al. / Phytochemistry 68 (2007) 591–595
purchased from Wako Pure Chemical Industries Ltd. Osaka, Japan (Wako 076-02841). The inhibition was measured spectrophotometrically at 37 °C using 1 mM p-nitrophenyl a-D-glucopyranoside, and p-nitrophenyl bD-glucopyranoside as a substrate at pH 6.9, then at pH 4.0 using 1 mM p-nitrophenyl a-D-mannopyranoside and 500 U/ml enzymes, in 50 mM sodium phosphate buffer containing 100 mM NaCl. 1-Deoxynojirimycin (0.425 mM) and acarbose (0.78 mM) were used as positive controls. The increment in absorption at 400 nm due to the hydrolysis of PNP-G by glycosidase was monitored continuously with the spectrophotometer (Molecular Devices, USA) (see Table 2). Acknowledgements We thank R. Machinek for the NMR spectra, H. Frauendorf for the mass spectra, and F. Lissy for microbiological work. H. M. P. P. is thankful to the DAAD (Deutscher Akademischer Austauschdienst) for a research grant J. D. W. thank the IFS (International Foundation of Sciences) for a providing financial support (F/3978-1). References Andersen, P.C., Gorbert, D.W., 2002. Influence of year and planting date on fatty acid chemistry of high oleic acid and normal peanut genotypes. J. Agric. Food Chem. 50, 1298–1305.
595
Ayafor, F.J., Ngadjui, B.T., Sondengam, B.L., Tsamo, E., 1984. A contribution to the phytochemistry of Zanthoxylum tessmannii. Planta Med. 50, 210–213. Chaaib, F., Queiroz, F.E., Ndjoko, K., Diallo, D., Hostettmann, K., 2003. Antifungal and antioxidant compounds from the root bark of Fagara zanthoxyloides. Planta Med. 69, 316–320. Cheung, H.T., Williamson, D.G., 1969. N.M.R. signals of methyl groups of triterpenes with oxygen functions at positions 2,3, and 23. Tetrahedron 25, 119–128. Gunasekera, S.P., Cordell, G.A., Farnsworth, N.R., 1982. Constituents of Pithecellobium multiflorum. J. Nat. Prod. 45, 651. Kerharo, J., Adam, J.G., 1971. La Pharmacope´e se´ne´galaise traditionnelle. Vigot Fre`res, Paris, pp. 715–723. Kumar, N.S., Muthukuda, M., Wazeer, M.I.M., 1985. A lupenediol from Euonymus revolutus. Phytochemistry 24, 1337–1340. Maskey, R.P., Asolkar, R.N., Kapaun, E., Wagner-Do¨bler, I., Laatsch, H., 2002. Phytotoxic arylethylamides from limnic bacteria using a screening with microalgae. J. Antibiot. 55, 643–649. Morris, R.J., McCartney, M.J., Bone, Q., 1984. The sterol chemistry of salps: the occurrence and distribution of saturated sterols. J. Marine Biol. Ass. United Kingdom 64, 343–349. Oki, T., Matsui, T., Osajima, Y., 1999. Inhibitory effect of a-glucosidase inhibitors varies according to its origin. J. Agric. Food Chem. 47, 550– 553. Raponda-Walker, A., Sillans, R., 1961. Plantes utiles du Gabon, Encyclopedie Biologique. P. Chevalier, Paris, 381–383. Razdan, T.K., Kachroo, P.K., Qurishi, M.A., Kalla, A.K., Waight, E.S., 1996. Unusual homologous long-chain alkanoic acid esters of lupeol from Koelpinia linearis. Phytochemistry 41, 1437–1438. Roitman, J.N., Jurd, L., 1978. Triterpenoid and phenolic constituents of Colubrina granulosa. Phytochemistry 17, 491–494. Shchukin, I.V., Medvedeva, S.A., 1999. Electrochemical oxidation of vanillic acid. Russ. J. Electrochem. 35, 599–601.
